Project Summary/Abstract Immunotherapies are revolutionizing oncology allowing many cancer patients with aggressive disease to enjoy durable remissions with excellent quality of life. However, these therapies are generally not effective for patients with cold solid tumors that have few infiltrating T cells and low levels of antigen presentation. IL-12 is a highly inflammatory cytokine with the potential to transform cancer immunotherapy. It can make cold tumors hot, drive elimination of tumors as a single agent and synergize with checkpoint inhibitors and adoptive cellular therapy in multiple experimental models. Despite multiple clinical trials of IL-12, using direct intravenous administration or a variety of formulations and vehicles, finding an optimal delivery method remains a critical barrier to its widespread clinical use. Several intra-tumor (IT) IL-12 gene delivery vehicles are currently being tested clinically; however each of these has major drawbacks that could prevent general implementation. Our group has been collaborating with the Seattle-based biotechnology company, Immune Design, to test two separate, novel and highly promising IL-12-producing vectors for gene delivery. One uses a potent self-replicating RNA delivery system to delivery high levels of IL-12 production through out the tumor. The other uses a lentivirus with an envelope that specifically targets dendritic cells. While both experimental vectors can eliminate tumors and prevent re-challenge in murine models, the significant investment required for clinical-grade virus production has impaired their swift movement into the clinic. Using a large animal model to demonstrate anti-tumor efficacy in a spontaneous cancer will catalyze the production of a clinical-grade product, thus in this proposal we will test both these vectors in spontaneous high grade soft tissue sarcomas in companion animals. If both vector approaches are efficacious, the analysis described here will help identify which vector should move towards testing in humans (using a human IL-12- containing construct). Furthermore, our comparison of these two agents will answer a fundamental question in cancer immunology; i.e., can precise delivery of IL-12 to DCs achieve superior anti-tumor immunity despite fewer transduced cells and a lower overall level of IL-12 production?